Biochemical and genetic characterization of trans-2'-carboxybenzalpyruvate hydratase-aldolase from a phenanthrene-degrading Nocardioides strain.
trans-2'-Carboxybenzalpyruvate hydratase-aldolase was purified from a phenanthrene-degrading bacterium, Nocardioides sp. strain KP7, and characterized. The purified enzyme was found to have molecular masses of 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography. Thus, the homotrimer of the 38-kDa subunit constituted an active enzyme. The Km and kcat values of this enzyme for trans-2'-carboxybenzalpyruvate were 50 microM and 13 s(-1), respectively. trans-2'-Carboxybenzalpyruvate was transformed to 2-carboxybenzaldehyde and pyruvate by the action of this enzyme. The structural gene for this enzyme was cloned and sequenced; the length of this gene was 996 bp. The deduced amino acid sequence of this enzyme exhibited homology to those of trans-2'-hydroxybenzalpyruvate hydratase-aldolases from Pseudomonas putida PpG7 and Pseudomonas sp. strain C18.
Iwabuchi
T
T
Marine Biotechnology Institute, Kamaishi Laboratories, Iwate, Japan.
Harayama
S
S
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D010616:Phenanthrenes / Q000378:metabolism*; D010667:Phenylpyruvic Acids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D009097:Multienzyme Complexes; D010616:Phenanthrenes; D010667:Phenylpyruvic Acids; D011994:Recombinant Proteins; C031181:phenanthrene; C037695:2'-carboxybenzalpyruvate; C110680:2'-carboxybenzalpyruvate hydratase-aldolase; D000446:Aldehyde-Lyases; D006836:Hydro-Lyases